Insecticides

The neonicotinyl insecticides evaluated were acetamiprid (Assail, Cerexagri, Inc.,  www.cerexagri.com), thiacloprid (Calypso, Bayer CropScience,  www.bayercropscience.com) and clothianidin (Clutch, Arvesta Corp.,  www.arvesta.com). Other insecticides were included in bioassays and field trials as standards; these are products typically used for pest control by apple growers in Washington or are new products that have a particular mode of activity that was of interest in this study. These included spinosad (Success, Dow AgroSciences LLC,  www.dowagro.com), methoxyfenozide (Intrepid, Dow AgroSciences LLC,), difluorobenzamide (Rimon, Crompton Corp.,  www.cromptoncorp.com), azinphosmethyl (Guthion, Bayer CropScience), and phosmet (Imidan, Gowan Company,  www.gowanco.com).

Rearing methods

Laboratory colonies of leafrollers were established from field populations collected from apple. The P. pyrusana colony was started from larvae collected in a commercial apple orchard near Yakima, Washington, in 1985, and the C. rosaceana colony from larvae collected in a commercial orchard near Mattawa, Washington, in 1990. Larvae were reared on an artificial pinto-bean diet following the methods of Shorey and Hale (1965). Colonies were maintained at constant temperature (23°C, L:D 16:8). The L. subjuncta colony originated from larvae collected in an apple orchard near Quincy, Washington, in 1999. Larvae were reared in the laboratory on a combination of artificial cutworm diet (Bio-Serv, #F9170,  www.bio-serv.com) and untreated apple leaves (Malus domestica Borkhausen, ‘Delicious’) following the methods described by Doerr et al. (2002). C. pomonella were obtained as eggs or pupae from a colony maintained at the USDA-ARS laboratory in Wapato, Washington, since 1960.

Dose-response bioassays (C. rosaceana, P. pyrusana and L. subjuncta neonates, apple)

A leaf disk bioassay was used to assess toxicity of the insecticides to neonate larvae. Treatments were prepared from a stock solution using formulated insecticide in 0.5 liter water. Four to eight concentrations were made by serial dilution of the stock solution. Two μl of a wetting agent (Latron B-1956, Dow AgroSciences), were added to each concentration. Each bioassay also included a check (water plus wetting agent). ‘Delicious’ apple leaves were collected from an orchard that had received no pesticide applications. Leaves were dipped in an insecticide solution three times to ensure adequate wetting and then allowed to air dry. A 2.3 cm diameter leaf disk was cut from each treated leaf, and four leaf disks treated with the same concentration of insecticide were placed in a small covered petri dish (Falcon 1006, 50 × 9 mm, Becton-Dickinson Labware,  www.bdbiosciences.com). Five 1- to 2-day-old larvae per petri dish were placed directly on the leaf disks. Ten dishes (50 larvae) were used for each insecticide concentration. Petri dishes were placed inside a plastic container and kept at constant temperature (23° C, L:D 16:8). Mortality was evaluated after 7 days. Failure to move one body length in response to probing with a fine camel's-hair brush was scored as dead.

Dose-response bioassay (C. pomonella neonates, apples)

An apple-dip bioassay was used to assess toxicity of the insecticides to neonate C. pomonella larvae. Treatments were prepared by diluting formulated insecticide in 1 liter water containing 0.32 ml/liter of an organosilicone surfactant (Sylwet L-77, Helena Chem. Co.,  www.helenachemical.com). Seven concentrations were made by serial dilution of the stock solution, with each bioassay having a check (water plus surfactant). Mature ‘Delicious’ apples were collected from an unsprayed orchard. Ten apples were dipped in each concentration and allowed to air dry. A section of waxed paper (2 × 2 cm) containing 10 C. pomonella eggs was placed on the stem end of the treated apples. The apples with eggs were placed individually in clear plastic containers (Anchor Packaging #409CX,  www.anchorpackaging.com) and kept in a growth chambers (22°C, L:D 16:8). The eggs hatched in approximately 4–5 days, and the number of successful larval entries was recorded at 14 days.

Dose-response bioassay (C. pomonella eggs, topical exposure)

A modification of the apple-dip bioassay described above was used to assess toxicity of insecticides to C. pomonella eggs when applied topically. Preparation of the concentrations and fruit treatment were as described above, except only six concentrations were used. Organic ‘Fuji’ apples free from insecticide residues were obtained from a packing house in Wenatchee, Washington. C. pomonella pupae were placed in a 60 × 30 × 36 cm wire mesh oviposition chamber. After C. pomonella adults emerged, 60 untreated apples were placed in the chamber, and females were allowed to lay eggs on them for 72 h. A carbohydrate and water source (5% honey dissolved in water) was introduced into the chamber using cotton wicking. Ten apples containing at least five eggs each were dipped into the same concentration of an insecticide, allowed to air dry, and then placed in clear plastic containers. The apples with C. pomonella eggs were held under constant conditions at 23° C, L:D 16:8. C. pomonella egg hatch was recorded after 10 days. Only eggs that showed no signs of larval development were recorded as dead; larvae that died while hatching were not.

Dose-response bioassay (C. pomonella eggs, residual exposure)

A modification of the apple-dip bioassay described above was used to assess toxicity of insecticide residues to C. pomonella eggs. Preparation of the concentrations, source of fruit, fruit treatment, and oviposition chambers were as described above. Treated apples (‘Fuji’), were placed in an oviposition chamber with C. pomonella adults, and females were allowed to oviposit for 24 hours. Apples were removed and placed individually in clear plastic containers, and held in growth chambers at 23° C, L:D 16:8. C. pomonella egg hatch was recorded after 10 days. Egg mortality was assessed as described above for the topical ovicide bioassay.

Dose-response bioassay (C. pomonella adults, residual exposure)

Treated plastic containers were used to evaluate the residual toxicity of insecticides to C. pomonella adults. Treatments were prepared by diluting formulated insecticide in 1 liter water containing 0.32 ml/liter of the organosilicone surfactant. Six or seven concentrations were used for each insecticide. One ml of an insecticide/surfactant mixture was added to a 120 ml plastic portion cup (Prairie Packaging, Inc., #S400,  www.prairiepack.com). The concentration in the cup was swirled for approximately 15 seconds, removed, and the residue allowed to air dry. Five cups (replicates) were treated per concentration. C. pomonella pupae were held in an untreated plastic emergence chamber, and adults allowed to emerge. Five 1- to 2-day-old unsexed C. pomonella adults were added to each of the treated cups. A carbohydrate and water source (5% honey dissolved in water) was introduced into the cup using cotton wicking. Cups were held at constant conditions of 23° C, L:D 16:8. Adult survival was assessed after 24 hours. A moth was considered dead if no response to probing with a camel's hair brush was observed.

Field-aged residue bioassays (C. rosaceana, P. pyrusana, L. subjuncta, C. pomonella neonates)

An evaluation of field-aged insecticide residues was conducted using modifications of the methods described above for dose-response bioassays. In each test, insecticides were applied to previously untreated ‘Delicious’ apple trees. Treatments were applied to the point of drip using a handgun sprayer at 300 psi to achieve thorough coverage. Plots with single trees, replicated three times, were used. An untreated check and, in most cases, an industry standard insecticide were included in each experiment. Ten leaves or 10 apples per replicate were collected from the interior canopy of each tree at 1, 4, 7, 14, 21 and 28 days after treatment. For C. rosaceana, P. pyrusana, and L. subjuncta, five arenas were prepared from each treated or check tree (15 arenas/treatment), and for C. pomonella 10 arenas were prepared for each treated or check tree (30 arenas/treatment). The bioassays were prepared, stored and evaluated as described above in the dose-response protocol (leaf-disk or apple-dip bioassays).

Data analysis of bioassays

Probit regression lines and LC₅₀ values were estimated using the probit option of POLO-PC (LeOra Software 1987). Probit models from each bioassay were then used to calculate toxicity indices relative to field use rates. Expected mortality was calculated using each probit model at 1× and 0.1× MFR (maximum field rate, the highest labeled concentration for use on apples, assuming a dilute spray). For the field-aged residue studies, mean mortality data were corrected by Abbott's formula (Abbott 1925), then analyzed using a one-way analysis of variance (SAS Institute 1995). A t ratio test on the slope parameter was used to determine if the data of each probit analysis fit a linear model (Robertson and Preisler 1992).

Field trials

Small plots, one to three trees in a single row, were sprayed with a handgun to the point of drip to obtain thorough coverage, simulating a dilute spray. Buffer trees and rows were included in the plot layout to ensure treatments did not contaminate neighboring plots. Handgun applications were made with a multiple-tank sprayer (Parker Mfg., Wenatchee, Washington).

Airblast plot sizes ranged from three trees (single row) to 15 trees (5 trees × 3 rows), with appropriate buffer trees and rows. A dual-tank airblast sprayer (Rears Pak-Blast, Rears Mfg., Eugene, OR) was calibrated to deliver either 234 or 935 liters/ha (see tables). Experimental orchards for all field trials were located in the vicinity of Wenatchee, Washington; the cultivar sampled was ‘Delicious’.

C. pomonella sprays targeted the first and second generations. The first application for each generation was based on a degree-day model (Beers et al. 1993) adapted from the principle described by Baskerville and Emin (1968). Subsequent applications for the generation were an interval of days based on the presumed length of residual control of the materials being evaluated. C. pomonella control was assessed by picking and examining 100 fruit per replicate just prior to commercial harvest (late August through mid-September), and the number of C. pomonella-injured fruits was recorded.

Field experiment design and analysis

Field experiments were randomized complete block designs, with 3–5 replications. C. pomonella fruit injury was expressed as a percentage reduction of injury from the untreated check [(1-(proportion injured fruit in treatment/proportion in check)) × 100]. Thus, a higher value indicated better C. pomonella control. This result was then compared to the industry standard organophosphate insecticide (Fisher's Least Significant Difference Test, P = 0.05) (SAS Institute 1995). In most cases the standard was azinphosmethyl, in one case it was phosmet, and in eight cases no organophosphate standard was included. In addition, a metadata analysis was done on the percentage reduction of fruit injury for acetamiprid and thiacloprid. A homogeneity of slopes model (PROC GLM, SAS institute 1995) was used to test the relationship between fruit injury and total g AI/ha applied, and whether or not the addition of oil affected the efficacy. F-tests were used to determine significance.

Larval bioassays

The LC₅₀ estimates for leafrollers to the neonicotinyl insecticides were high relative to that of spinosad, the most commonly used insecticide for leafroller control in the post-bloom period (NASS 2002) (Table 1). The toxicity indices for both P. pyrusana or C. rosaceana at 1× MFR were estimated to be less than 70%, while at 0.1 × MFR the indices for all three neonicotinyl insecticides were estimated to be equal to or less than 1%. For spinosad, the toxicity index at only 0.1 × MFR was estimated to be above 95% mortality for both C. rosaceana and P. pyrusana. The toxicity index is not a direct predictor of an insecticide's field efficacy, as other factors such as longevity of residues and interactions with plant surfaces can mediate performance. The toxicity index can be useful in comparing insecticides within a bioassay technique, using field rates for standardization. Further, if the bioassay method used reflects to a reasonable degree an insect stage's exposure to a pesticide in the field, the toxicity index of a candidate insecticide can be used as an indicator of the inherent toxicity in comparison to a compound with known field performance characteristics, e.g., spinosad.

L. subjuncta results were similar to those for leafrollers. Although only two neonicotinyl insecticides were tested both had toxicity indices of less than 30% mortality for the 1 × MFR and less than 1% mortality for the 0.1 × MFR (Table 1). Relative to spinosad, none of the neonicotinyl insecticides have high inherent toxicity to leafrollers or L. subjuncta, and would therefore be less likely to provide control under field conditions.

The LC₅₀ estimates for the neonicotinyl insecticides against C. pomonella larvae were small relative to those for leafrollers or L. subjuncta, but were similar to the LC₅₀ for azinphosmethyl (Table 1). The toxicity indices at 1 × MFR were above 94% mortality for the neonicotinyl insecticides, while that of azinphosmethyl was essentially 100% (the probit model asymptotes as it approaches 100%). At 0.1 × MFR, the estimated C. pomonella larval mortality was between 60–70% for the neonicotinyl insecticides, while the toxicity index for azinphosmethyl at 0.1 × MFR was higher, nearly 98%. Data from these bioassays indicate that the neonicotinyl insecticides are not as toxic to C. pomonella larvae as the industry standard, azinphosmethyl.

Ovicide bioassays (C. pomonella)

The method of exposure affected the toxicity of neonicotinyl insecticides to C. pomonella eggs. Acetamiprid and thiacloprid had LC₅₀s similar to the insect growth regulators when eggs were exposed topically (Table 2). The toxicity indices of acetamiprid and thiacloprid (topical exposure) at 1 × MFR were similar to the insect growth regulators (methoxyfenozide and difluorobenzamide), while clothianidin was lower. At 0.1 × MFR, acetamiprid and thiacloprid had values slightly lower than the insect growth regulators, with clothianidin substantially lower at 8% estimated egg mortality (Table 2). These data support the premise that two of the neonicotinyl insecticides, acetamiprid and thiacloprid, would have some topical activity against C. pomonella eggs, however, not to the same degree as the insect growth regulators.

The residual exposure method produced much higher LC₅₀s for C. pomonella eggs for all three neonicotinyl insecticides compared to the topical exposure method (Table 2). The toxicity indices at 1 × MFR for the neonicotinyl insecticides were only 33–53% mortality, whereas the indices for the insect growth regulators were >97%. At 0.1 × MFR, the toxicity indices for the neonicotinyl insecticides were <25% mortality compared to 82% and 54% mortality for methoxyfenozide and difluorobenzamide, respectively (Table 2).

The two insect growth regulators showed little differences in activity against C. pomonella eggs based on the route of exposure, and both would be expected to be effective in killing C. pomonella eggs, at least as fresh residues. Acetamiprid and thiacloprid would be expected to have some effect on C. pomonella eggs present at the time of an application, but less effect on eggs laid thereafter. Clothianidin would not be expected to have much effect on C. pomonella eggs. The combination of limited ovicidal activity and higher larvicidal activity may enhance field performance of acetamiprid and thiacloprid against C. pomonella.

Adult bioassays (C. pomonella)

The neonicotinyl insecticides had less effect on adult C. pomonella overall than azinphosmethyl, as estimated by the toxicity index (Table 3). Clothianidin and acetamiprid had high toxicity indices at 1 × MFR, while at 0.1 × MFR all of the neonicotinyl insecticides had low toxicity indices relative to azinphosmethyl. Adulticidal activity of the neonicotinyl insecticides would likely be expected to provide limited enhancement to overall C. pomonella control.

Field-aged residue bioassays

The residual activity of neonicotinyl insecticides against P. pyrusana and C. rosaceana reflected their low toxicity in dose-response bioassays. While acetamiprid had some activity against P. pyrusana and C. rosaceana as 1-day-old residues, its activity dropped off quickly thereafter (Table 4). At 7 days (P. pyrusana) or 21 days (C. rosaceana), mortality was not different from the untreated check. Thiacloprid and clothianidin had less activity than acetamiprid, with 1- and 4-day-old residues not causing higher mortality than the untreated check. None of the neonicotinyl insecticides demonstrated residual activity similar to that of the standard, spinosad, which caused high levels of mortality through 21 days. While these data cannot be directly compared because the tests were not conducted within the same year, it appears that the neonicotinyl insecticides would not provide sufficient efficacy against leafrollers under field conditions.

Acetamiprid was the only neonicotinyl insecticide tested against L. subjuncta. There was virtually no activity of this material against L. subjuncta larvae even on 1-day-old residues (Table 4).

All three neonicotinyl insecticides produced high levels of mortality of C. pomonella neonates for at least 14 d, and had substantial activity even after 28 days (Table 4). Clothianidin has a shorter length of residual activity, based on the 21 day evaluation. The standard insecticide, azinphosmethyl, also showed high levels of activity through 21 days, with some residual activity even after 28 days. Twenty-one days has been a standard re-treatment interval for azinphosmethyl use for C. pomonella control for many years (Smith et al. 2004), and is inevitably the standard reapplication period against which new materials are compared.

Thiacloprid was the only neonicotinyl insecticide tested in the field-aged residue bioassay examining ovicidal activity against C. pomonella eggs. Residual activity was short and variable compared to the insect growth regulators (Table 4), which caused >85% egg mortality after 28 days. However, the level of egg mortality in the field-aged bioassay was higher than might have been expected based on laboratory bioassays of C. pomonella eggs laid on fresh residues of thiacloprid (Table 2). In the residual C. pomonella egg bioassay, thiacloprid had the least activity of the neonicotinyl insecticides and C. pomonella egg mortality of more than 80% as observed in the field-aged residue bioassay would not have been predicted. It is possible that thiacloprid residues in the apple-dip bioassay method used in the laboratory were much lower than those that resulted from the field applications. While the laboratory method of dipping apples in insecticide concentrations with a wetting agent ensured uniform coverage of fruit, the addition of the wetting agent could have resulted in lower levels of thiacloprid per unit of surface area compared to those deposited by the field application method. While thiacloprid, and possibly the other two neonicotinyl insecticides, might provide some mortality of C. pomonella eggs laid on residues, the levels of mortality are not sufficient to warrant shifting timing away from the primary target, the neonate larvae.

The laboratory and field-aged residue bioassays described above provide a foundation for understanding how new insecticides proposed for the apple pest control system function against lepidopteran pests. Using these techniques, candidate insecticides can be screened rapidly and inexpensively against different pests and different life stages of those pests. When compared with insecticides known to be effective against a pest or particular pest stage, the relative toxicities of candidate insecticides can be characterized through dose-response bioassays. Further, because these tests are conducted with known susceptible populations that are maintained under controlled conditions, reference data are also available for use in future comparisons with field-collected populations when resistance issues arise. The field-aged residue bioassays also provide insights into the longevity of candidate insecticides' residual activities and can help establish re-treatment intervals for initial field trials.

In this study with neonicotinyl insecticides, bioassay data from both laboratory and field experiments helped eliminate the need and expense of conducting field trials against leafrollers and L. subjuncta. Further, bioassays revealed the toxicity of neonicotinyl insecticides to C. pomonella eggs when applied topically, indicating that they are more than just larvicides. This may help explain why the neonicotinyl insecticides are more effective management tools than might be expected from their larvicidal activity alone. Separating the effects of an insecticide on insect life stages using bioassays also provides clues as to how to best target more susceptible stages and time applications in field trials. This information is difficult to obtain from field trials without greatly increasing sample time and intensity, because pest life stages typically overlap during the course of a treatment regime.

C. pomonella field trials, efficacy

Acetamiprid provided a moderate to high degree of fruit protection in all field trials over five years (Table 5), with an average of 82.2% (range 59–95%) reduction in percentage C. pomonella fruit injury when compared to the untreated check. The relative percentage reduction in fruit damage was positively related (F = 4.03, P = 0.05, R² = 0.13) to the total g AI/ha applied (Fig. 1). Although some of the treatments included a 235 g AI/ha rate, the current label limits the rate to 167 g AI/ha. The addition of oil to acetamiprid did not affect the percentage relative control (F = 0.15, P= 0.71).

Fruit protection from thiacloprid was more variable than acetamiprid in all field trials over four years, with an average of 71.0% (range 43–97%) reduction in percentage fruit injury compared to the untreated check. The greater variation compared to acetamiprid was in part due to the lower rates of thiacloprid that were used in some trials. The highest levels of control achieved (97%) were in treatments where eight applications were made (1,800 to 2,394 g AI/ha/year); this exceeds the amount allowed by the current label (maximum of 280 g AI/ha per application; 560 g/ha/ year). The level of control with the higher rates (>260 g AI/ha) was generally better than at lower rates, but still varied from 58–72% control for four applications. The effect of total g AI/ha applied significantly affected the percentage control of C. pomonella (F = 8.53, P = 0.01, R² = 0.39), however, the addition of oil did not (F = 1.71, P = 0.21) (Fig. 2).

Although fewer trails were performed with clothianidin, results suggest that it is less effective than the other two neonicotinyl insecticides. Relative reduction in percentage fruit injury averaged 28% (range 5–49%) (Table 5). This lower level of control in field trials would not be predicted if only the results from the larval bioassay were considered, where clothianidin was similar to acetamiprid and thiacloprid. However, bioassay results for toxicity to adults, eggs, and length of residual control of larvae were all lower for clothianidin, and the combination of reduced effects from all routes of exposure was likely sufficient to adversely affect field performance.

In all field trials where the mean percentage of injured fruit in a neonicotinyl insecticide treatment was significantly different from the organophosphate standard (azinphosmethyl or phosmet), control with the neonicotinyl insecticides was always inferior (Table 5). The level of control as measured by percentage fruit injury with azinphosmethyl averaged 94.2% over all trials (data not shown). In the absence of organophosphate insecticide resistance, azinphosmethyl is still the most effective insecticide tested against C. pomonella. The field trial results are consistent with the high toxicity indices in larval bioassays, as well as the long residual control in field-aged residue bioassays with azinphosmethyl.